Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium

Abstract

Background: Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been used successfully for optical mapping in cardiac cells and tissues. However, their utility for probing electrical activity deep inside the myocardial wall and in blood-perfused myocardium has been limited because of light scattering and high absorption by endogenous chromophores and hemoglobin at blue-green excitation wavelengths.

Objective: The purpose of this study was to characterize two new styryl dyes--di-4-ANBDQPQ (JPW-6003) and di-4-ANBDQBS (JPW-6033)--optimized for blood-perfused tissue and intramural optical mapping.

Methods: Voltage-dependent spectra were recorded in a model lipid bilayer. Optical mapping experiments were conducted in four species (mouse, rat, guinea pig, and pig). Hearts were Langendorff perfused using Tyrode's solution and blood (pig). Dyes were loaded via bolus injection into perfusate. Transillumination experiments were conducted in isolated coronary-perfused pig right ventricular wall preparations.

Results: The optimal excitation wavelength in cardiac tissues (650 nm) was >70 nm beyond the absorption maximum of hemoglobin. Voltage sensitivity of both dyes was approximately 10% to 20%. Signal decay half-life due to dye internalization was 80 to 210 minutes, which is 5 to 7 times slower than for di-4-ANEPPS. In transillumination mode, DeltaF/F was as high as 20%. In blood-perfused tissues, DeltaF/F reached 5.5% (1.8 times higher than for di-4-ANEPPS).

Conclusion: We have synthesized and characterized two new near-infrared dyes with excitation/emission wavelengths shifted >100 nm to the red. They provide both high voltage sensitivity and 5 to 7 times slower internalization rate compared to conventional dyes. The dyes are optimized for deeper tissue probing and optical mapping of blood-perfused tissue, but they also can be used for conventional applications.

Figure 1

Structure of the new NIR styryl dyes JPW-6003 (Di-4-ANBDQPQ), and JPW-6033 (Di-4-ANBDQBS). Structure of di-4-ANEPPS is shown for comparison. Grey square shows the chromophores, and the white dashed square is a linker.

Figure 2

Spectral characteristics of JPW-6003 and JPW-6033. A, B.: Dye absorbance and emission spectra in ethanol. C, D.:Voltage-sensitive transmission ΔT/T and fluorescence excitation ΔF/F spectra in the hemispherical lipid bilayer for 100 mV voltage steps. The fluorescence was collected through a >715 nm long-pass filter. For comparison, the voltage-sensitivity (ΔF/F) in pig heart derived from optical action potentials at 520 and 650 nm are shown (black squares for JPW-6003, white squares for JPW-6033).

Figure 3

Optical action potentials (OAP) recorded in different experimental models using JPW-6003 and JPW-6033. Gray and black traces show raw and processed recordings, respectively. A.: Epifluorescence recordings in Tyrode’s-perfused preparations of pig, guinea pig, rat, and mouse. B.: Epifluorescence recordings in blood-perfused Langendorff pig (Recordings were made from LV). Right OAP was recorded using di-4-ANEPPS. C.: Transillumination recordings in Tyrode’s-perfused pig (RV).

Figure 4

Washout kinetics of JPW-6003 and JPW-6033 in rat (Tyrode’s perfusion). Panels A and B show the time dependence of the total (F) and voltage-sensitive fluorescence (ΔF/F), respectively. Zero on the time scale corresponds to the maxima. Vertical lines show the times corresponding to 50% drop from the maximum value. Labels next to the lines show the half-life. The kinetics of the dye di-4-ANNEPPS is shown for comparison. All curves are averages of 3–5 experiments, and error bars show the level of data variation.

Figure 5

Dye washout kinetics in pig (Tyrode’s perfusion). Notations are the same as in Figure 4. To obtain the half-life, the curves were extrapolated (dotted lines).

Figure 6

Loading and washout kinetics of JPW-6003 in blood-perfused pig heart (direct injection into blood flow). A.: Time dependence of total F and voltage-sensitive fluorescence ΔF/F. t=0 corresponds to the end of dye injection. F is normalized to maximum. B.: Responses of F and ΔF/F to a switch from blood- to Tyrode’s-perfusion. F is normalized to the stationary value. The switch was performed after 120 minutes of blood-perfusion (during stationary stage). Dashed lines show extrapolation of the blood-perfusion curves. All curves with error bars are averages of 3–5 experiments. Thick black and grey lines near time axis indicate switching between blood- and Tyrode’s-perfusion, respectively.

Figure 7

Dye loading and washout kinetics in blood-perfused pig heart with accelerated (Tyrode’s) loading. t=0 corresponds to the end of dye injection. The switch from Tyrode’s- to blood-perfusion at 14 minutes after dye injection. A.: The kinetics of total fluorescence F (normalized to maximum value). B.: Voltage-sensitive fluorescence ΔF/F. Notations are the same as in Figure 6.

Figure 8

Stability of electrophysiological characteristics after injection of JPW-6003 and JPW-6033. A.: Volume-conducted ECG recorded in an isolated blood-perfused porcine heart before and 30 min after injection of 8.7 μmol JPW-6033 (50 nmol/g tissue). B.: Time dependence of conduction velocity (CV) in guinea pig during and after injection of 220 nmol JPW-6033 (100 nmol/g tissue). Bottom plot shows dose vs. time. C.: Time dependence of action potential duration (APD) in rat after injection of 160 nmol JPW-6003 (100 nmol/g tissue). Plot shows data from 3 experiments (different symbols). The trend (solid line) shows slight increase (0.8 ms/hr).